The long range-goal is to understand the mechanism and extent of allelic exclusion (random monoallelic expression) in the CNS, and to determine whether such expression may contribute to the puzzling inheritance pattern observed in some CNS disorders including schizophrenia, bipolar disorder, and multiple sclerosis. Allelic exclusion in the CNS has until now been difficult to study because differences in replication timing between alleles, a key epigenetic component of monoallelic expression, can only be studied in dividing cells, and also because analysis of random allele-specific expression usually requires single-cell assays. Recent advances allowing derivation of clonal cell lines from neural stem cells (NSCs) in culture should thus provide a major advantage for the systematic analysis of allele-specific replication timing and allelic exclusion in the CNS. We propose to use these cells to test the following specific hypothesis: Is there enrichment for allelically excluded genes adjacent to known clusters of allelically excluded odorant receptor (OR) genes? The specific aims are to determine 1) if clonal NSC lines can be used as a model system to study allele-specific expression and replication timing, and 2) if genes adjacent to OR gene clusters at schizophrenia susceptibility loci are subject to allelic exclusion. We will use clonal lines from NSCs cultured from FI Mus musculus C57BL/6 x Mus musculus molossinus hybrids, which will allow the analysis of any gene showing at least a single base difference between the two strains. The cells will be assayed directly for DNA replication timing; they may also be differentiated into neuronal or astrocytic populations for the analysis of allele-specific expression. The genes will be analyzed by means of quantitative RT-PCR and PCR assays previously developed in this laboratory. The results will reveal whether there is domain-wide clustering of allelic exclusion, as hypothesized, a first step in understanding the signals involved. Furthermore, any monoallelically expressed genes discovered may be relevant to schizophrenia. Finally, positive results would indicate that the approach can be used as a tool for the discovery of allelically excluded genes in other chromosomal regions relevant to other similarly inherited CNS disorders. The results of this study may thus help us to identify the genes that cause schizophrenia and other genetic disorders of the CNS. [unreadable] [unreadable] [unreadable] [unreadable]